Our long term objective is to understand the fundamental principles underlying skin morphogenesis. In the main grant, "Development and Regeneration of Skin Appendages" (AR 42177, 1/1/2004-12/31/2008), we hypothesized that the "competence" of epidermal cells to form different types of skin appendages is gradually restricted during development in the process of "commitment". While classical tissue interaction experiments have defined competence and commitment with many interesting examples, their molecular bases remain mostly unknown. Recent progress in epigenetics suggests that epigenetic changes on the chromatin not only play a role in cancer, but may also have a role in stem cell progression. Our preliminary data suggest their involvement in skin development. Here we hypothesize that epigenetic changes in chromatin play a central role in maintaining or restricting the multi-potentiality of epidermal precursor cells, therefore contributing to the molecular mechanisms of competence and commitment, respectively. To test this hypothesis, we will evaluate the expression of chromatin modulation enzymes such as DMA methyltransferase, histone methyltransferases, histone deacetylases during skin appendage development, and test their functional involvement with inhibitors to these enzymes in the skin reconstitution explant cultures. To search for candidate target genes, we will use the newly available chicken microarrays to profile changes during skin development with or without inhibitors to chromatin modulation enzymes. Homeobox containing genes will be our priority. As an example we will use ChIP analyses and methylation specific PCR to follow the methylation status of the Msx 2 promoter. This proposal represents the application of current technology and a novel concept to answer fundamental questions raised 40 years ago. It has great potential to break into new levels of understanding in development mechanisms of the skin. It may generate new approaches on how to guide stem cells in the context of tissue engineering. We hope you can expedite the approval of this relatively small supplement so we can evaluate these exciting possibilities promptly. Based on that, we may then develop a full scale study. [unreadable] [unreadable]